Glut4-mediated glucose uptake represents the rate-limiting step of insulin-stimulated glucose disposal, and type 2 diabetes is associated with impaired translocation of Glut4 to the cell surface. The insulin- responsive pool of Glut4 is localized in intracellular membrane vesicles, or IRVs, that deliver Glut4 to the plasma membrane. Proteomics analyses performed in our and other laboratories have shown that, in addition to Glut4, these vesicles contain two major proteins, IRAP and sortilin. The challenge now is to understand how these seemingly unrelated proteins find each other in the cell and form a unique type of a vesicular carrier. Based on the results obtained in the previous funding period, we put forward a hypothesis that may explain the process of the IRV formation. According to our model, the cytoplasmic tails target the major IRV proteins to the perinuclear donor membranes, where Glut4 and IRAP interact with sortilin via lumenal domains. The heteromeric complex consisting of Glut4, IRAP and sortilin is distributed from the donor membranes to the IRV as a single entity with the help of GGA and ACAP-1 adaptors, small GTPases of the Arf family and phosphatidylinositol phosphates. In Specific Aim 1, we will test this hypothesis and further characterize the role of lumenal interactions in protein targeting into the IRVs. In Specific Aim 2, we will determine the biological functions of the individual IRV component proteins. In Specific Aim 3, we will explore the actual mechanism of the IRV formation and, in particular, determine the role of cytoplasmic sortilin-binding proteins, Arf6 and PIP4 in this process. PUBLIC HEALTH RELEVANCE: Impaired translocation of Glut4 to the cell surface may represent a primary factor for the development of type 2 diabetes mellitus. Recent studies point out to the cell biology of Glut4 recycling as a potential site of primary diabetes-related abnormalities. We propose to explore several unknown aspects of Glut4 traffic which may shed light on the molecular nature of insulin resistance and diabetes.